Device and method for rapid tissue preparations for histological investigations

ABSTRACT

The device ( 1 ) for fast tissue processing for histological examination consists of a housing ( 2 ) that is covered by a transparent hood ( 70 ). A baseplate ( 3 ) divides the device ( 1 ) into an upper part ( 4   a ) and a lower part ( 4   b ). Several process chambers that are served by a transport mechanism ( 6 ) with holders ( 40 ) for tissue specimens are provided for on the baseplate ( 3 ). At least one reservoir ( 74 ) is provided for in the lower part ( 4   b ) of the device, which has a flow connection to at least one process chamber. An operating panel ( 76 ) allows the user to enter the appropriate data.

The invention relates to a device for fast tissue processing forhistological examinations, with a housing that is divided into an upperpart and a lower part, several process chambers that are arranged on thebaseplate, and a transport mechanism that transfers the tissue samplesto the process chambers.

In addition, the invention relates to a method for fast tissueprocessing for histological examinations.

Document WO 01/44783 discloses a fast tissue processor. A microwavegenerator is attached to a reaction chamber for the tissue. Themicrowave generator produces warmth that allows the processes which thetissue samples undergo to run more quickly. For processing the tissuesamples, several different modules that contain various liquids ofvarying composition are provided for. A disadvantage is that warmingwith microwaves may have an effect on the tissues being prepared.

U.S. Pat. No. 5,560,956 discloses a tissue processor that consists ofnumerous liquid reservoirs. The liquids in the reservoirs are pumpedinto a process chamber. After processing, the liquid is pumped back intothe reservoir. A chamber for wax is also provided for. The preparationprocess provided for with this device takes approximately 8 to 12 hours,which cannot be considered a fast tissue processing.

Another tissue processor is disclosed in U.S. Pat. No. 3,771,490. Here,a sample chamber is provided for, into which the samples to be processedare placed. The various liquids are pumped into the sample chamber fromchambers located beneath it. A disadvantage is that the various reagentsmust be introduced into a sample chamber, which leads to rapidcontamination of the reagents. This makes frequent changing of thereagents necessary.

The Leica TP 1020 is a tissue processor with several process chambersthat are arranged in a circle. The reagents for the tissue samples arecontained in the process chambers. Two wax-filled chambers, which can beheated, are also provided for. The mechanism for changing the tissuesamples is disclosed in German Patent DE 196 47 662. A mechanism todrive the tissue processor that processes samples for histologicalexamination is provided for beneath the process chambers. A centrallylocated guide rod is moved by means of a toothed belt. The guide rod maybe raised or lowered by a movement of the toothed belt via the drivemechanism. A carrousel is attached to the upper area of the guide rod,which has several object holders that are introduced into one of theprocess chambers. Each of the object holders has a lid with acircumferential seal. After the holder is introduced into one of theprocess chambers, they are rendered completely airtight by the seal.

The underlying problem of the invention is to create a device in whichsmall tissue samples (biopsies) can be prepared quickly and economicallyfor histological preparation. In the process, the risks associated withthe reagents used are to be substantially reduced.

This problem is resolved by a device with the features described in thecharacterizing part of claim 1.

A further problem of the invention is to develop a method by which smalltissue samples (biopsies) can be processed quickly and economically forhistological preparation.

This problem is resolved by a method that is characterized by the stepsin claim 14.

It is of advantage for the process of desiccation and infiltration withparaffin of small volumes of tissue sample (such as biopsies) to bereduced to only a few steps with a short dwell time. An example would bea dwell time of 30 seconds in ethanol; a dwell time of 6 minutes inacetone; and a dwell time of 6 minutes in paraffin. Here, the entireprocess takes 12.5 minutes. Because this process must be started severaltimes per day for urgent testing, a capacity of 20 cassettes issuitable. A small table device should allow for these circumstances. Inorder to optimize desiccation in spite of the short dwell time, thereagents (ethanol and acetone) are exchanged via a reservoir, incontrast to known tissue processors. This reservoir has many times thevolume of the process chambers that contain the preparations. Thiscircumstance has several advantages. In spite of the small size of theprocess chamber, a large volume of liquid is available. The processchamber can be emptied and refilled with fresh reagent during theprocess. A certain number of processes can be carried out with supplyfrom a freshly filled reservoir, without having to refill it with freshreagent. This is also the case when the used reagent is pumped back intothe reservoir.

Emptying the process chamber after each step of the process has beenconcluded also has the advantage that all the reagent, which is usuallya volatile solvent, is back in the reservoir. The solvents that are usedpose a certain safety risk (flammability, fumes). This emptying alsoreduces the handling of these solvents.

The device is advantageously fitted with a housing that is divided by abaseplate into an upper part and a lower part. Several process chambersare arranged on the baseplate, and a transport mechanism transfers thetissue samples into the process chambers. The process chamber itself hasa first opening through which the process chamber may be filled oremptied. Several reservoirs containing reagent are provided for in thehousing beneath the baseplate, such that the reagent may be pumped fromone of the reservoirs into the process chamber and from the processchamber back into that reservoir.

The process chambers are designed in the form of cups, each of which hasa side wall and a floor, such that the first opening and the secondopening are in the floor of the process chamber. This is an advantagebecause the tubing that feeds the process chambers with reagent leadsdirectly from the floor of the process chamber in the bottom part of thedevice. As a result, there is no clutter of tubing in the upper part ofthe device.

The transport mechanism is fitted with a transport arm to which isattached a holder for several cassettes with histological tissuesamples. A lid is attached to the holder, which closes the processchamber when the process chamber is in the lowered holder. The lid has acertain freedom of movement, which, when the holder is shaken, allows itto remain seated on the process chamber.

The subject of the invention is schematically depicted in the diagram,and will be described in the following figures. They show:

FIG. 1 a schematic diagram of the arrangement of the process chambers onthe baseplate inside the device;

FIG. 2 a schematic diagram of the arrangement of the process chambers,showing the tissue carriers lowered into the last process chamber.

FIG. 3 the drive and movement mechanism to raise and lower the holderwith the tissue carriers into and out of the process chamber;

FIG. 4 a schematic perspective diagram of the holder;

FIG. 5 a lateral view of a cassette for receiving histological tissuesamples;

FIG. 6 a schematic perspective diagram of a reservoir;

FIG. 7 a perspective diagram of the device; and

FIG. 8 a schematic diagram of a process chamber connected to the pumpand a reservoir; and

FIG. 9 a further development of the arrangement of process chambers,pump, and reservoir, in which the process chamber exhibits two openings.

An arrangement of several process chambers (11, 12, 13, 14, and 15) on abaseplate (3) is shown in FIG. 1 and FIG. 2. A schematic perspective ofthe entire device (1) for the fast processing of tissue samples forhistological examination is shown in FIG. 7. In the embodiment depictedin FIG. 1, the process chambers are labeled 11, 12, 13, 14, and 15. Alinear arrangement of the process chambers is also conceivable. A device(1) for the fast processing of tissue samples for histologicalexamination consists of a housing (2), which the baseplate (3) separatesinto an upper part (4 a) and a lower part (4 b). The followingdescription is limited to a circular arrangement of the process chambers(11, 12, 13, 14, and 15). However, this should not be viewed as alimitation of the invention.

In the center (5) of the baseplate (3), a transport mechanism (6) with atransport arm (7) are provided for in the housing (2) of the device (1).The device (1) is designed to carry out a process of desiccation andsubsequent infiltration with paraffin (wax) of small sample volumes.Small sample volumes such as biopsies are placed in the process chambers(11, 12, 13, 14, and 15) of the device (1) so the water may be withdrawnfrom them using various solvents. Replacement of this water (tissuefluid) is then done in other process chambers using paraffin (wax).These process chambers are designed to be heatable. The tissue samplesthemselves are placed in special cassettes (50, see FIG. 5). Severalcassettes (50) are used in a common holder (40), which is lowered intoor lifted out of the various process chambers by means of the transportmechanism (6). There is room for approximately 20 cassettes (50) in theholder (40), which can be manufactured out of, among other things,plastic. The plastic manufacture of the holder (40) allows it to be forsingle-use. Once the process has run its course, that is, the tissuesample has been desiccated and the water replaced with paraffin, theholder (40) does not need to be cleaned for the next round ofprocessing. The used holder is disposed of or recycled. A new holder(40) is used for each round of processing.

To transport the holder (40) from one process chamber to the nextprocess chamber, a relative motion is generated between the transportmechanism (6) and the baseplate (3). In the embodiment described here,the baseplate (3) is stationary and the transport mechanism (6) movesfrom process chamber to process chamber.

In the embodiment depicted in FIG. 1 and FIG. 2, six positions (P₁, P₂,P₃, P₄, P₅, and P₆) are provided for, of which five positions (P₁, P₂,P₃, P₄, and P₅) are each fitted with a process chamber. Each processchamber is in the form of a cup (8) that has a cylindrical side wall (9)that is closed on one side by a floor (10). The process chamber itselfmay have either one or two openings. In process chambers that have oneopening, filling and emptying occur through this one opening, which runsinto the floor (10) of the process chamber. In process chambers with twoopenings, a constant irrigation of the process chamber with solvent ispossible. Good irrigation is achieved when one opening is situated onthe floor and the second opening at the top of the fluid level in theprocess chamber. In the device according to the invention, filling,emptying, and irrigation are done only in those process chambers intowhich solvent is to be introduced.

In the embodiment depicted in FIG. 1, the first process chamber (11) isprovided for in position P₁. This first process chamber (11) representsthe loading position of the device (1). For example, a holder (40) withcassettes (50) for histological processing can be placed in the processchamber (11). A liquid suitable for medium-term storage and to preventdrying of the tissue samples is contained in the first process chamber(11). The liquid (fixans) in the first process chamber (11) may beformaldehyde, for example. A second process chamber (12) follows thefirst process chamber (11) in position P₂. The holder (40) is raisedsomewhat and attached to the transport arm (7) of the transportmechanism (6), which is situated above the first process chamber (11).When the process starts, the transport arm (7) lifts the holder (40) andlowers it into the second process chamber (12). In the meantime theholder (40) is transferred to the second process chamber (12), andliquid is introduced into the second process chamber (12) through anopening in the second process chamber (12). The liquid used is ethanol.Filling should be done quickly in order to prevent drying of the tissuesamples. While the holder (10) is in the second process chamber (12),the holder (10) is moved up and down (agitated) to optimize irrigationof the tissue samples with liquid. The holder (40) remains in the secondprocess chamber (12) for time t₁. Time t₁ in the process chamber (12),which is filled with ethanol, should not exceed 30 seconds.

A third process chamber (13) follows the second process chamber (12) inposition P₃. The holder (40) is lifted out of the second process chamber(12) with the transport arm (7) attached to the transport mechanism (6),and transported to the third process chamber (13). While the holder (40)is transferred to the third process chamber (13), liquid is introducedinto the third process chamber (13) through an opening in the floor (10)of the third process chamber (13). The liquid used is acetone. Thesecond process chamber (12) is emptied in parallel during this time. Atthe least, the second and third process chambers (12 and 13) are fittedwith one fluid pump each (not depicted). The directionality of the fluidpumps is reversible. As a result, liquid can be pumped into and out of aprocess chamber without the necessity of valves. The holder (40) remainsin the third process chamber (13) for a time, t₂, such that time t₂ doesnot exceed six minutes.

A fourth process chamber (14) that is filled with wax is provided for inposition P₄. A fifth process chamber (15) that is also filled with waxis provided for in position P₅. Two wax baths (wax-filled processchambers) are commonly used, because the wax in the fourth processchamber (14), which is the first wax bath, generally becomescontaminated by solvent residue. At least one wax-filled process chambercontains no opening and is heatable. The wax chambers (two units areappropriate) must be filled manually. The holder (40) remains in one ofthe filled process chambers (14 and 15) for time t₃. Time t₃ in thewax-filled process chambers (14 and 15) should not exceed six minutes.However, it is conceivable to use only one wax-filled process chamber.In that case, the holder (40) remains in the single wax-filled processchamber for time t₃.

Position P₆ is empty and is designed as an unloading station. Theoperator can remove the holder (40) along with the prepared tissuesamples from the device (1). Position P₆ is fitted with a drip pan (16)for removing the preparation holder. For example, the transport arm (7)of the transport mechanism (6) removes the holder (40) from the fifthprocess chamber (15) and sets it on the drip pan (16). FIG. 2 depictsthe situation in which the holder has been lowered into the fifthwax-filled process chamber (15). The fifth process chamber (15) isclosed with a lid (17). The lid (17) is attached to the transport arm(7) of the transport mechanism (6) above the holder (40) which, as shownin FIG. 2, closes the process chamber into which the holder (40) islowered.

FIG. 3 shows the drive and motion mechanism (30) for lifting andlowering the holder (40) with the tissue carriers into the processchambers. The rest of the description relates to the device (1) with acircular arrangement of process chambers. The drive and motion mechanism(30) consists of a spindle (32) which serves to lift and lower thetransport arm (7). In the case of the central arrangement of thetransport mechanism (6), a steering mechanism (33) is provided foraround the spindle (32), which exhibits beveled surfaces (34)corresponding to the number of process chambers. The beveled surfaces(34) are each interrupted by gaps (35). When the transport arm (7) iscompletely lifted by the spindle (32), that is, the holder (40) has beencompletely lifted out of the process chamber, the transport arminterfaces with the surface (34). The transport arm (7) glides along thebeveled surface (34) in the direction of a gap (35) solely as a resultof gravity. The transport arm then drops through the gap (35) into theprocess chamber.

FIG. 4 shows a schematic perspective of a holder (40) for cassettes (seeFIG. 5). The holder consists of a first and a second rectangular part(41 and 42), which are both firmly attached to each other. Both thefirst and the second part (41 and 42) contain numerous shelves (43) thatare suitable for receiving cassettes. There are openings (46) in boththe floor (44) and the top cover (45) of the holder (40), as well as inthe individual shelves (43), which are designed for good irrigation ofthe holder (40) with reagent. Openings (48) in the side walls (47) ofthe holder (40) serve the same purpose. A bracket (49) is attached tothe top cover of the holder (40), which the transport arm (7) of thetransport mechanism (6) can grab hold of.

FIG. 5 is a lateral view of a cassette (50) for receiving a histologicaltissue sample and then processing it with the appropriate liquids. Thecassettes (50) can be used in the holder (40) shown in FIG. 4. Thecassette (50) consists of a floor (51) with attached side parts (59);the cassette (50) can be closed with a detachable top cover (52). Thereare numerous slits (53) in both the floor (51) and in the top cover (52)that ensure irrigation of the entire cassette (50) with liquid. The topcover (52) has several clasps (54) that engage with other clasps (55)that are arranged along the side parts (59) and the floor (51). Forbetter handling when opening and/or closing, brackets (56) are providedfor on the top cover (52).

FIG. 6 is a schematic perspective diagram of an embodiment of areservoir (60). The coupling of the reservoir (60) to the device (1) isachieved by means of a plug connector (61). The first reservoir opening(62) and second reservoir opening (63) in the reservoir (60) are locatedabove the fluid level (64). The first reservoir opening (62) is theoutflow of a channel (65) that reaches all the way to the floor (66) ofthe reservoir (60). The second reservoir opening (63) sticks out of in apart of the device (1) that is attached to suction. Fumes that develop,particularly when pumping the liquid back into the reservoir (60), arethus sucked out. When docking the reservoir (60) to the lower part (4 b)of the device (1), the reservoir opening (62) is tightly connected tothe pump via an O-ring (not depicted).

FIG. 7 again shows the entire device (1) in perspective. The upper part(4 a) of the device (1) is covered by a transparent hood (70). The hood(70) is set around the central transport mechanism (6) and consists of afirst part (71) and a second part (72) that may be opened and closedaround the central transport mechanism (6). The directionality ofopening and closing is indicated by the double arrows A-A and B-B. Foropening or closing, a latch (73) is attached to both the first part (71)and to the second part (72). Under the transparent hood (70), which ismade of plastic, may be seen the individual process chambers, thetransport arm (7) with the holder (40) for the cassettes (50), and thetransport mechanism (6). A fan with an activated charcoal filter (notdepicted), which draws off solvent fumes from the area of the hood (70)and directs it to an exhaust (not depicted) by means of a hose (notdepicted), is integrated into the device (1). The hood (70) coversalmost the entire baseplate (3) and the upper part (4 a) of the device(1). A first reservoir (74) and a second reservoir (75) for the solventsused in the device (1) are each integrated laterally into device in thelower part (4 b). The solvents may, for example, be ethanol and acetone.The first reservoir (74) and the second reservoir (75) may be removedfor refilling or to replenish the used solvent. At the front of thedevice, an operating panel (76) to start the program that runs thedevice (1) is depicted schematically. The operating panel (76) has adisplay (77) and several operator controls (78) by which the processingof tissue samples may be individually programmed. In addition, thefollowing undepicted elements are included in the area between the firstreservoir (74) and the second reservoir (75) in the lower part (4 b) ofthe device: two fluid pumps, the fan, the electrical drive mechanism forthe transport mechanism (6), a power source, and a controller.

FIG. 8 shows a schematic diagram of a process chamber (13) connected toa pump (80) and a reservoir (60). A first tube (82) leads from the firstreservoir opening (62) to the pump (80), and a second tube (83) leadsfrom the pump (80) to a first opening (81) in the floor (10) of theprocess chamber (13). The directionality of the pump (80) is reversible.Because of this, only one pump is needed to fill or empty the processchamber (13).

A further embodiment of the system consisting of a process chamber (13),a reservoir (60), and a pump (80) is depicted in FIG. 9. A first tube(82) leads from the first reservoir opening (62) to the pump (80), and asecond tube (83) from the pump (80) to an opening (81) in the floor (10)of the process chamber (13). There is a second opening (91) in theprocess chamber (13) that is connected with a third tube (92). Thesecond opening (91) may also the set in the floor (10) of the processchamber (13). The third tube (92) terminates at the second reservoiropening (63). This development makes it possible to continuouslycirculate the reagent between the reservoir (60) and the process chamber(13).

1. Device for fast tissue processing for histological examination, witha housing (2), which is divided into an upper part (4 a) and a lowerpart (4 b) by a baseplate (3); several process chambers (11, 12, 13, 14,15, 16), which define a side wall (9) and a floor (10), and are arrangedon the baseplate (3) and may be filled with a reagent; a transportmechanism (6), which transfers the tissue samples to the processchambers (11, 12, 13, 14, 15, 16), characterized in that the transportmechanism is (6) provided with a transport arm (7) to which a holder(40) is attached for several cassettes (50) for histological tissuesamples; in that the transport arm (7) is lowered by gravity therebyintroducing the holder (40) into the process chamber; and in that atleast one process chamber exhibits a first opening (81) in the side wall(9) or in the floor (10) by which the process chamber may be filled oremptied with reagent.
 2. Device according to claim 1, characterized inthat a lid (17) is attached to the top of the holder (40) such that whenthe holder (40) is lowered into the process chamber, the process chamberis closed.
 3. Device according to claim 2, characterized in that the lid(17) has a certain freedom of movement, so that, when the holder (40) isshaken in the process chamber by the transport mechanism (6), the lid(17) remains seated on the process chamber.
 4. Device according to claim1, characterized in that the transport mechanism (6) is situated in thecenter (5) of the baseplate (3) and the process chambers are arranged onthe baseplate (3) around the central transport mechanism (6).
 5. Deviceaccording to claim 1, characterized in that the process chambers arearranged linearly on the baseplate (3).
 6. Device according to claim 1,characterized in that at least one process chamber is provided with asecond opening (91), which together with the first opening (81) makespossible continuous pumping of reagent through the process chamber. 7.Device according to claims 1 through 6, characterized in that at leastone process chamber is designed with no opening and is heatable. 8.Device according to claim 1, characterized in that at least one processchamber is designed with no opening and is manually filled with wax. 9.Device according to claim 1, characterized in that one drive mechanism(30) is provided for, which causes a relative movement between thetransport mechanism (6) and the process chambers (11, 12, 13, 14, 15,16); and in that the lowering of the holder (40) into the processchamber is accomplished by gravity.
 10. Device according to claim 1,characterized in that the process chambers (11, 12, 13, 14, 15, 16) aredesigned in the form of a cup with a side wall (9) and a floor (10), andin that the first opening (81) and the second opening (91) are builtinto the floor (10) of the process chamber.
 11. Device according toclaim 10, characterized in that the volume of the process chambers (11,12, 13, 14, 15, 16) is a multiple of the volume of tissue samplecontained in the holder (40).
 12. Device according to claim 11,characterized in that the holder (40) manufactured out of plastic and isdesigned for single-use processing.
 13. Device according to claim 1,characterized in that several replaceable reservoirs (74, 75) forreagent are provided for under the baseplate (3) in the housing (2) ofthe device (1), such that the reagent may be pumped from one of thereservoirs (74, 75) into the process chamber and from the processchamber back into that reservoir (74, 75).
 14. Method for fastprocessing of histological tissue samples in several process chambers(11, 12, 13, 14, 15, 16), which define a side wall (9) and a floor (10),characterized by the following steps: lowering of a holder (40) withcassettes for histological processing into a loading position, wherebythe loading position consists of a first process chamber (11) in which areagent for intermediate storage of the tissue samples is provided for;lifting of the holder (40) by means of a transport arm (7) attached to atransport mechanism (6); transfer of the holder (40) to a second processchamber (12), such that during the intervening time, the second processchamber (12) is filled with reagent through an opening (81) in the floor(10) of the second process chamber (12). lowering of the holder (40)into the second process chamber (12), such that the holder (40) remainsthere for time t₁; removal of the holder (40) and transfer of the holder(40) to a third process chamber (13), such that the second processchamber (12) is emptied during the intervening time, and the thirdprocess chamber (13) is filled; lowering of the holder (40) into thethird process chamber (13), such that the holder (40) remains there fortime t₂; removal of the holder (40) from the third process chamber (13),such that the third process chamber (13) is emptied after removal of theholder (40); lowering of the holder (40) into a wax-filled processchamber (14), such that the holder remains there for time t₃; andlowering of the holder into an unloading station.
 15. Method accordingto claim 14, characterized in that time t₁ in the second process chamber(12), which is filled with the reagent ethanol, does not exceed 30seconds.
 16. Method according to claim 14, characterized in that time t₂in the third process chamber (13), which is filled with the reagentacetone, does not exceed 6 minutes.
 17. Method according to claim 14,characterized in that time t₃ in the wax-filled process chamber (14)does not exceed 6 minutes.
 18. Method according to claim 17,characterized in that another wax-filled process chamber (114) isdownstream from the wax-filled process chamber (14) in order to reducetransfer and contamination with solvent.
 19. Method according to claim14, characterized in that the process chambers are filled and emptiedfrom and into one of the replaceable reservoirs (74, 75), and in whichthe reservoirs (74, 75) are provided for in a lower part (4 b) of thehousing (2), and in which the lower part (4 b) is separated from anupper part (4 a) by a baseplate (3).
 20. Method according to claim 14,characterized in that a relative movement is generated between theprocess chambers (11,12,13,14,15,16) and the transport arm (7) in orderto achieve transfer of the holder (40) from one process chamber to thenext process chamber.